Clathrin-coated vesicles (CCVs) play a fundamental role in controlling a cell's interaction with its environment. They are responsible for receptor-mediated endocytosis and the intracellular targeting of molecules during the biogenesis of organelles of the endocytic and regulated secretory pathway. Thus CCV function is critical for cellular metabolism of nutrients, processing of antigens, control of growth factor receptor expression and hormonal secretion. Understanding the molecular basis for cellular control of CCV formation therefore has implications in treating such diverse illnesses as heart disease, infection, cancer and diabetes. Furthermore, CCVs are the prototype coated vesicle involved in intracellular membrane transport. Hence, understanding regulation of CCV formation provides insight into general mechanisms of molecular traffic between cellular membranes and organelles. The driving force for CCV formation is self-assembly of the protein clathrin, a process that must be regulated in the cell. Work from past funding of this project has defined the biochemical properties of clathrin and the cellular role of CCVs. During the next funding period we will continue to focus on how intrinsic features of clathrin and their interaction with extrinsic factors regulate CCV formation and function. We propose four specific aims for the next funding period, investigating functional features of the clathrin heavy chain and light chain subunits. The experiments planned involve interplay between in vitro systems, cellular assays and genetic manipulation and are as follows. Aim 1 is to establish how clathrin heavy chain phosphorylation regulates CCV function and to investigate the hypothesis that this modification regulates cross-talk between receptor signaling in rafts and CCV-mediated receptor endocytosis. Aim 2 is to establish the functions mediated by the distal and terminal domains of the clathrin heavy chain in regulating CCV assembly. Aim 3 is to characterize proteins interacting with different domains of the two clathrin light chains (LCa and LCb) expressed in vertebrates and to study the function of LC-binding proteins at the cellular level. Aim 4 is to establish the differential regulatory functions of the clathrin light chains LCa and LCb at the level of the vertebrate organism by genetic manipulation of zebrafish and mice. We anticipate that these latter studies will initiate new approaches to investigation of human disease states in which clathrin function is involved.